In vivo and in vitro analyses of the role of the Prc protease in inducing mucoidy in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, alginate biosynthesis gene expression is inhibited by the transmembrane anti-sigma factor MucA, which sequesters the AlgU sigma factor. Cell envelope stress initiates cleavage of the MucA periplasmic domain by site-1 protease AlgW, followed by further MucA degradation to release AlgU. However, after colonizing the lungs of people with cystic fibrosis, P. aeruginosa converts to a mucoid form that produces alginate constitutively. Mucoid isolates often have mucA mutations, with the most common being mucA22, which truncates the periplasmic domain. MucA22 is degraded constitutively, and genetic studies suggested that the Prc protease is responsible. Some studies also suggested that Prc contributes to induction in strains with wild-type MucA, whereas others suggested the opposite. However, missing from all previous studies is a demonstration that Prc cleaves any protein directly, which leaves open the possibility that the effect of a prc null mutation is indirect. To address the ambiguities and shortfalls, we reevaluated the roles of AlgW and Prc as MucA and MucA22 site-1 proteases. In vivo analyses using three different assays and two different inducing conditions all suggested that AlgW is the only site-1 protease for wild-type MucA in any condition. In contrast, genetics suggested that AlgW or Prc act as MucA22 site-1 proteases in inducing conditions, whereas Prc is the only MucA22 site-1 protease in non-inducing conditions. For the first time, we also show that Prc is unable to degrade the periplasmic domain of wild-type MucA but does degrade the mutated periplasmic domain of MucA22 directly. IMPORTANCE: After colonizing the lungs of individuals with cystic fibrosis, Pseudomonas aeruginosa undergoes mutagenic conversion to a mucoid form, worsening the prognosis. Most mucoid isolates have a truncated negative regulatory protein MucA, which leads to constitutive production of the extracellular polysaccharide alginate. The protease Prc has been implicated, but not shown, to degrade the most common MucA variant, MucA22, to trigger alginate production. This work provides the first demonstration that the molecular mechanism of Prc involvement is direct degradation of the MucA22 periplasmic domain and perhaps other truncated MucA variants as well. MucA truncation and degradation by Prc might be the predominant mechanism of mucoid conversion in cystic fibrosis infections, suggesting that Prc activity could be a useful therapeutic target.

Exploration of the tunability of BRD4 degradation by DCAF16 trans-labelling covalent glues.

Chemically induced proximity modalities such as targeted protein degradation (TPD) hold promise for expanding the number of proteins that can be manipulated pharmacologically. However, current TPD strategies are often limited to proteins with preexisting ligands. Molecular glues (e.g. glutarimide ligands for CUL4CRBN), offer the potential to target undruggable proteins. Yet, their rational design is largely unattainable due to the unpredictability of the 'gain-of-function' nature of the glue interaction upon chemical modification of ligands. We recently reported a covalent trans-labelling glue mechanism which we named 'Template-assisted covalent modification', where an electrophile decorated BRD4 inhibitor was effectively delivered to a cysteine residue on DCAF16 due to an electrophile-induced BRD4-DCAF16 interaction. Herein, we report our efforts to evaluate how various electrophilic modifications to the BRD4 binder, JQ1, affect DCAF16 recruitment and subsequent BRD4 degradation efficiency. We discovered a moderate correlation between the electrophile-induced BRD4-DCAF16 ternary complex formation and BRD4 degradation. Moreover, we show that a more solvent-exposed warhead presentation optimally recruits DCAF16 and promotes BRD4 degradation. The diversity of covalent attachments in this class of BRD4 degraders suggests a high tolerance and tunability for the BRD4-DCAF16 interaction. This offers a new avenue for rational glue design by introducing covalent warheads to known binders.

Post-Artesunate Delayed Hemolysis in Pediatric Malaria Patients in the United States.

Post-artesunate delayed hemolysis (PADH) occurred in 6 of 24 children treated with artesunate for severe malaria in the United States; however severe hemolysis requiring hospitalization or transfusion was rare. In children in the U.S. treated with artesunate, counseling and symptom monitoring may be preferred to weekly laboratory surveillance for PADH.

Template-assisted covalent modification underlies activity of covalent molecular glues.

Molecular glues are proximity-inducing small molecules that have emerged as an attractive therapeutic approach. However, developing molecular glues remains challenging, requiring innovative mechanistic strategies to stabilize neoprotein interfaces and expedite discovery. Here we unveil a trans-labeling covalent molecular glue mechanism, termed 'template-assisted covalent modification'. We identified a new series of BRD4 molecular glue degraders that recruit CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). Through comprehensive biochemical, structural and mutagenesis analyses, we elucidated how pre-existing structural complementarity between DCAF16 and BRD4BD2 serves as a template to optimally orient the degrader for covalent modification of DCAF16Cys58. This process stabilizes the formation of BRD4-degrader-DCAF16 ternary complex and facilitates BRD4 degradation. Supporting generalizability, we found that a subset of degraders also induces GAK-BRD4BD2 interaction through trans-labeling of GAK. Together, our work establishes 'template-assisted covalent modification' as a mechanism for covalent molecular glues, which opens a new path to proximity-driven pharmacology.

Optical torque calculations and measurements for DNA torsional studies.

The angular optical trap (AOT) is a powerful instrument for measuring the torsional and rotational properties of a biological molecule. Thus far, AOT studies of DNA torsional mechanics have been carried out using a high numerical aperture oil-immersion objective, which permits strong trapping but inevitably introduces spherical aberrations due to the glass-aqueous interface. However, the impact of these aberrations on torque measurements is not fully understood experimentally, partly due to a lack of theoretical guidance. Here, we present a numerical platform based on the finite element method to calculate forces and torques on a trapped quartz cylinder. We have also developed a new experimental method to accurately determine the shift in the trapping position due to the spherical aberrations by using a DNA molecule as a distance ruler. We found that the calculated and measured focal shift ratios are in good agreement. We further determined how the angular trap stiffness depends on the trap height and the cylinder displacement from the trap center and found full agreement between predictions and measurements. As a further verification of the methodology, we showed that DNA torsional properties, which are intrinsic to DNA, could be determined robustly under different trap heights and cylinder displacements. Thus, this work has laid both a theoretical and experimental framework that can be readily extended to investigate the trapping forces and torques exerted on particles with arbitrary shapes and optical properties.

Epidemiology and risk stratification of young infants presenting to the emergency department with hypothermia.

Objective: Hypothermic infants are presumed to be at high risk for a serious bacterial infection (SBI) or herpes simplex virus (HSV) infection. In contrast to febrile infants, the emergency department (ED) management of hypothermic infants is variable in the absence of consensus guidelines, potentially resulting in low-value care and missed diagnoses. We investigated the diagnostic workup conducted for hypothermic infants in our academic pediatric ED, the incidence of SBI and HSV infection, and risk factors associated with infection. Methods: We conducted a single-center retrospective study of infants ≤90 days of age with a rectal temperature ≤36.5°C in the ED between 2013 and 2022. From their medical records, we abstracted the type(s) of testing each infant received in the ED and the diagnosis of SBI and HSV, analyzing characteristics associated with each. Results: Of 1095 hypothermic infants identified, 402 (37%) underwent testing for SBI or HSV. Among these, 34/402 (8.5%) had an SBI or HSV. A minimum temperature below 36°C and hospital admission were characteristics associated with higher rates of infectious testing. Infants aged 29‒90 days, compared to 0‒28 days, were more likely to have a urinary tract infection (odds ratio 3.28, 95% confidence interval 1.47‒7.32). Conclusions: Hypothermic infants have slightly lower rates of SBI or HSV than febrile infants, for whom infectious studies are widely recommended, but still high enough to warrant an infectious workup in most cases. Further research is required to risk stratify hypothermic infants in the ED to standardize care and improve outcomes while optimizing resource utilization.

The effects of Asian American Pacific Islander (AAPI) data inequities in gynecologic oncology.

Asian American and Pacific Islanders (AAPI) are the fastest growing racial group in the United States. Data on AAPI communities, however, are significantly limited. The oversimplification and underreporting of this ethnically and socioeconomically heterogenous population through the use of aggregated data has deleterious effects and worsens disparities in patient treatment, outcomes, and experiences. Gynecologic oncology disparities do not exist in a vacuum, and are rooted in larger cultural gaps in our understanding and delivery of healthcare. In this paper, we aim to demonstrate how AAPI data inequities have negative downstream effects on research and public health policies and initiatives, and also provide a call to action with specific recommendations on how to improve AAPI data equity within these realms.

Caudal regression in fetus with de novo SMARCA2 pathogenic variant.

Nicolaides-Baraitser syndrome (NCBRS) is a rare autosomal dominant genetic condition that is characterized by severe intellectual disability, dysmorphic facial features, short stature, sparse hair, and early onset seizures. This diagnosis is established by suggestive clinical findings and the identification of a heterozygous SMARCA2 pathogenic variant by molecular genetic testing. There are not, however, consensus clinical diagnostic criteria for this condition as there are so few documented cases. Here, we present a case of prenatally diagnosed caudal regression with sacral agenesis and congenital vertical talus (rocker bottom feet) that was ultimately found to have a de novo SMARCA2 pathogenic variant. The patient had an amniocentesis with normal karyotype and microarray followed by failed direct rapid whole exome sequencing (WES) due to maternal cell contamination. She elected for termination of the pregnancy based on the clinical prognosis of the ultrasound findings; WES revealed a pathogenic variant after her termination. We believe this is the first case of these findings associated with NCBRS. If any future cases of either finding are found in association with a SMARCA2 genetic variant, caudal regression and rocker bottom feet should be included in the spectrum of physical traits associated with this pathogenic variant.

In vivo and in vitro analysis of the role of the Prc protease in inducing mucoidy in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, alginate biosynthesis gene expression is inhibited by the transmembrane anti-sigma factor MucA, which sequesters the AlgU sigma factor. Cell envelope stress initiates cleavage of the MucA periplasmic domain by site-1 protease AlgW, followed by further MucA degradation to release AlgU. However, after colonizing the lungs of people with cystic fibrosis, P. aeruginosa converts to a mucoid form that produces alginate constitutively. Mucoid isolates often have mucA mutations, with the most common being mucA22 , which truncates the periplasmic domain. MucA22 is degraded constitutively, and genetic studies suggested that the Prc protease is responsible. Some studies also suggested that Prc contributes to induction in strains with wild type MucA, whereas others suggested the opposite. However, missing from all previous studies is a demonstration that Prc cleaves any protein directly, which leaves open the possibility that the effect of a prc null mutation is indirect. To address the ambiguities and shortfalls, we reevaluated the roles of AlgW and Prc as MucA and MucA22 site-1 proteases. In vivo analyses using three different assays, and two different inducing conditions, all suggested that AlgW is the only site-1 protease for wild type MucA in any condition. In contrast, genetics suggested that AlgW or Prc act as MucA22 site-1 proteases in inducing conditions, whereas Prc is the only MucA22 site-1 protease in non-inducing conditions. For the first time, we also show that Prc is unable to degrade the periplasmic domain of wild type MucA, but does degrade the mutated periplasmic domain of MucA22 directly. IMPORTANCE: After colonizing the lungs of individuals with cystic fibrosis, P. aeruginosa undergoes mutagenic conversion to a mucoid form, worsening the prognosis. Most mucoid isolates have a truncated negative regulatory protein MucA, which leads to constitutive production of the extracellular polysaccharide alginate. The protease Prc has been implicated, but not shown, to degrade the most common MucA variant, MucA22, to trigger alginate production. This work provides the first demonstration that the molecular mechanism of Prc involvement is direct degradation of the MucA22 periplasmic domain, and perhaps other truncated MucA variants as well. MucA truncation and degradation by Prc might be the predominant mechanism of mucoid conversion in cystic fibrosis infections, suggesting that Prc activity could be a useful therapeutic target.

Optical Torque Calculations and Measurements for DNA Torsional Studies.

The angular optical trap (AOT) is a powerful instrument for measuring the torsional and rotational properties of a biological molecule. Thus far, AOT studies of DNA torsional mechanics have been carried out using a high numerical aperture oil-immersion objective, which permits strong trapping, but inevitably introduces spherical aberrations due to the glass-aqueous interface. However, the impact of these aberrations on torque measurements is not fully understood experimentally, partly due to a lack of theoretical guidance. Here, we present a numerical platform based on the finite element method to calculate forces and torques on a trapped quartz cylinder. We have also developed a new experimental method to accurately determine the shift in the trapping position due to the spherical aberrations by using a DNA molecule as a distance ruler. We found that the calculated and measured focal shift ratios are in good agreement. We further determined how the angular trap stiffness depends on the trap height and the cylinder displacement from the trap center and found full agreement between predictions and measurements. As further verification of the methodology, we showed that DNA torsional properties, which are intrinsic to DNA, could be determined robustly under different trap heights and cylinder displacements. Thus, this work has laid both a theoretical and experimental framework that can be readily extended to investigate the trapping forces and torques exerted on particles with arbitrary shapes and optical properties. SIGNIFICANCE: We developed a simulation platform based on the finite element method for force and torque calculation for particles in an angular optical trap (AOT), with considerations of tightly focused Gaussian beam, spherical aberrations, and optically anisotropic particles. Experimental measurements of focal shift ratio, force, and torque under multiple conditions were in good agreement with predictions from the simulations. We also demonstrated that intrinsic DNA torsional properties can be robustly measured under different AOT measurement conditions, strongly validating our simulations and calibrations. Our platform can facilitate trapping particle design for single-molecule assays using the AOT.

Diagnosis and Management of an Enlarging Placental Immature Teratoma: A Case Report.

The clinical imaging and pathology of a rare case of immature teratoma of the placenta is presented with a discussion of controversies related to classification and clinical suggestions for therapy and follow-up.

Adipocytes Are the Only Site of Glutamine Synthetase Expression Within the Lactating Mouse Mammary Gland.

Background: Glutamine in milk is believed to play an important role in neonatal intestinal maturation and immune function. For lactating mothers, glutamine utilization is increased to meet the demands of the enlarged intestine and milk production. However, the source of such glutamine during lactation has not been studied. Objectives: We aimed to assess the effects of lactation on the expression of glutamine synthetase (GS) in the mammary gland and other tissues of lactating mice. Methods: Mouse tissues were sampled at 4 time points: 8-wk-old (virgin, control), post-delivery day 5 (PD5, early lactation), PD15 (peak lactation), and involution (4 days after weaning at PD21). We examined the gene expression and protein concentrations of GS and the first 2 enzymes of branched-chain amino acid catabolism: branched-chain aminotransferase 2 (BCAT2) and branched-chain ketoacid dehydrogenase subunit E1α (BCKDHA). Results: The messenger RNA (mRNA) expression and protein concentrations of GS in mammary glands were significantly lower at PD5 and PD15 compared with the control but were restored at involution. Within the mammary gland, GS protein was only detected in adipocytes with no evidence of presence in mammary epithelial cells. Compared with the control, mRNA and protein concentrations of BCAT2 and BCKDHA in mammary glands significantly decreased during lactation and involution. No changes in GS protein concentrations during lactation were found in the liver, skeletal muscle, and lung. In non-mammary adipose tissue, GS protein abundance was higher during lactation compared with the virgin. Conclusions: This work shows that, within the mouse mammary gland, GS is only expressed in adipocytes and that the relative GS abundance in mammary gland sections is lower during lactation. This suggests that mammary adipocytes may be a site of glutamine synthesis in the lactating mouse. Identifying the sources of glutamine production during lactation is important for optimizing milk glutamine concentration to enhance neonatal and maternal health.

Use of a Large Language Model to Assess Clinical Acuity of Adults in the Emergency Department.

Importance: The introduction of large language models (LLMs), such as Generative Pre-trained Transformer 4 (GPT-4; OpenAI), has generated significant interest in health care, yet studies evaluating their performance in a clinical setting are lacking. Determination of clinical acuity, a measure of a patient's illness severity and level of required medical attention, is one of the foundational elements of medical reasoning in emergency medicine. Objective: To determine whether an LLM can accurately assess clinical acuity in the emergency department (ED). Design, Setting, and Participants: This cross-sectional study identified all adult ED visits from January 1, 2012, to January 17, 2023, at the University of California, San Francisco, with a documented Emergency Severity Index (ESI) acuity level (immediate, emergent, urgent, less urgent, or nonurgent) and with a corresponding ED physician note. A sample of 10 000 pairs of ED visits with nonequivalent ESI scores, balanced for each of the 10 possible pairs of 5 ESI scores, was selected at random. Exposure: The potential of the LLM to classify acuity levels of patients in the ED based on the ESI across 10 000 patient pairs. Using deidentified clinical text, the LLM was queried to identify the patient with a higher-acuity presentation within each pair based on the patients' clinical history. An earlier LLM was queried to allow comparison with this model. Main Outcomes and Measures: Accuracy score was calculated to evaluate the performance of both LLMs across the 10 000-pair sample. A 500-pair subsample was manually classified by a physician reviewer to compare performance between the LLMs and human classification. Results: From a total of 251 401 adult ED visits, a balanced sample of 10 000 patient pairs was created wherein each pair comprised patients with disparate ESI acuity scores. Across this sample, the LLM correctly inferred the patient with higher acuity for 8940 of 10 000 pairs (accuracy, 0.89 [95% CI, 0.89-0.90]). Performance of the comparator LLM (accuracy, 0.84 [95% CI, 0.83-0.84]) was below that of its successor. Among the 500-pair subsample that was also manually classified, LLM performance (accuracy, 0.88 [95% CI, 0.86-0.91]) was comparable with that of the physician reviewer (accuracy, 0.86 [95% CI, 0.83-0.89]). Conclusions and Relevance: In this cross-sectional study of 10 000 pairs of ED visits, the LLM accurately identified the patient with higher acuity when given pairs of presenting histories extracted from patients' first ED documentation. These findings suggest that the integration of an LLM into ED workflows could enhance triage processes while maintaining triage quality and warrants further investigation.

"Should We Phenobarb-it-All?" A Phenobarbital-Based Protocol for Non-Intensive Care Unit Trauma Patients at High Risk of or Experiencing Alcohol Withdrawal.

BACKGROUND: Alcohol use is frequent in trauma patients and alcohol withdrawal syndrome (AWS) is associated with significant morbidity. Benzodiazepines are commonly used for AWS, but may cause neurologic and respiratory adverse events (AEs). The objective was to evaluate the effectiveness and safety of a phenobarbital-based protocol for the treatment of AWS in non-intensive care unit (ICU) trauma patients. METHODS: Adult non-ICU trauma patients at high risk of or experiencing AWS PRE and POST implementation of a phenobarbital-based protocol were included. Outcomes were AWS-related complications (AWS-RC), benzodiazepine use, adjunctive medication use, hospital length of stay (HLOS), and medication-related AEs. Subgroup analyses were performed on patients with traumatic brain injury (TBI), rib fractures, and at high risk of severe AWS. RESULTS: Overall, 110 patients were included (51 PRE, 59 POST). AWS-RC developed in 17 PRE patients compared to 10 POST patients (33% vs 17%; P = .05). PRE patients were more likely to receive benzodiazepines (88% vs 42%, P < .0001) and higher total dose (11 vs 4 mg lorazepam equivalent; P = .001). No difference noted in HLOS (8 vs 8 days, P = .27), adjunctive medication use (49% vs 54%, P = .60), or AEs (57% vs 39%, P = .06). There was no difference in AWS-RC in the TBI subgroup (P = .19), less AEs in the rib fracture POST subgroup (P = .04), and less AWS-RC in the high risk of severe AWS POST subgroup (P = .03). DISCUSSION: A phenobarbital-based protocol in trauma patients is effective in preventing AWS-RC and decreasing benzodiazepine use without increasing AEs.

P. aeruginosa CtpA protease adopts a novel activation mechanism to initiate the proteolytic process.

During bacterial cell growth, hydrolases cleave peptide cross-links between strands of the peptidoglycan sacculus to allow new strand insertion. The Pseudomonas aeruginosa carboxyl-terminal processing protease (CTP) CtpA regulates some of these hydrolases by degrading them. CtpA assembles as an inactive hexamer composed of a trimer-of-dimers, but its lipoprotein binding partner LbcA activates CtpA by an unknown mechanism. Here, we report the cryo-EM structures of the CtpA-LbcA complex. LbcA has an N-terminal adaptor domain that binds to CtpA, and a C-terminal superhelical tetratricopeptide repeat domain. One LbcA molecule attaches to each of the three vertices of a CtpA hexamer. LbcA triggers relocation of the CtpA PDZ domain, remodeling of the substrate binding pocket, and realignment of the catalytic residues. Surprisingly, only one CtpA molecule in a CtpA dimer is activated upon LbcA binding. Also, a long loop from one CtpA dimer inserts into a neighboring dimer to facilitate the proteolytic activity. This work has revealed an activation mechanism for a bacterial CTP that is strikingly different from other CTPs that have been characterized structurally.

Group B Streptococcus Infection and Obstetric Hemorrhage Risk.

OBJECTIVE: To elucidate the association between GBS infection and maternal risk for obstetric hemorrhage (OBH) and OBH-related morbidities (OBH-M). METHODS: This was a retrospective cohort study of all deliveries with a documented GBS status at a single large academic medical center from 2018 to 2019. GBS status was determined by either urine culture or rectovaginal culture collected during the antepartum period. The primary outcomes were quantitative blood loss (QBL), OBH, and a composite of OBH-M. Secondary outcomes were individual components of the OBH-M composite and frequency of hemorrhage-related interventions utilized intrapartum and postpartum. A stratified analysis was conducted examining only patients who were diagnosed intrapartum with an intrapartum intraamniotic infection (III). RESULTS: Of 4679 pregnant individuals who delivered a live infant between January 1, 2018 and January 1,2019 with a documented GBS status, 1,487 were identified as GBS positive (+) and 3192 were identified as GBS negative (-). The GBS + group did not have significantly higher QBL (p = 0.29) or rate of OBH (p = 0.35). There were no significant differences by GBS status in OBH morbidity (p = 0.79) or its individual components or frequency of individual pharmacologic or non-pharmacologic OBHrelated interventions. There were also no significant differences by GBS status among patients with an III. CONCLUSIONS FOR PRACTICE: GBS infection at the time of delivery was not associated with increased risk for OBH or OBH-M. Further research is needed to further explore the relationship between peripartum infections and OBH risk.

Characterisation of digital therapeutic clinical trials: a systematic review with natural language processing.

Digital therapeutics (DTx) are a somewhat novel class of US Food and Drug Administration-regulated software that help patients prevent, manage, or treat disease. Here, we use natural language processing to characterise registered DTx clinical trials and provide insights into the clinical development landscape for these novel therapeutics. We identified 449 DTx clinical trials, initiated or expected to be initiated between 2010 and 2030, from ClinicalTrials.gov using 27 search terms, and available data were analysed, including trial durations, locations, MeSH categories, enrolment, and sponsor types. Topic modelling of eligibility criteria, done with BERTopic, showed that DTx trials frequently exclude patients on the basis of age, comorbidities, pregnancy, language barriers, and digital determinants of health, including smartphone or data plan access. Our comprehensive overview of the DTx development landscape highlights challenges in designing inclusive DTx clinical trials and presents opportunities for clinicians and researchers to address these challenges. Finally, we provide an interactive dashboard for readers to conduct their own analyses.